Pharmaceutical lipid compositions

ABSTRACT

The present invention relates to a particulate composition containing; a) 5 to 90% of at least one phosphatidyl choline component b) 5 to 90% of at least one diacyl glycerol component, at least one tocopherol, or mixtures thereof, and c) 1 to 40% of at least one non-ionic stabilizing amphiphile, where all parts are by weight relative to the sum of the weights of a+b+c and where the composition contains particles of at least one non-lamellar phase structure or forms particles of at least one non-lamellar phase structure when contacted with an aqueous fluid. The invention additionally relates to pharmaceutical formulations containing such compositions, methods for their formation and methods of treatment comprising their administration.

This application claim priority to PCT Application No.PCT/GB2005/004745, filed Dec. 9, 2005, which claims priority to GB0501364.4, filed. Jan. 1, 2005 and GB 0507812.6, filed Apr. 18, 2005,all of which are herein incorporated by reference in their entirety.

The present invention relates to the protection, solubilisation,stabilisation and delivery of active agents in pharmaceutical andneutraceutical compositions. In particular, the invention relates toamphiphilic compositions and formulations, and active agent deliverysystems based upon these.

Amphiphile-based formulations show considerable potential in thedelivery of many substances, especially for in vivo delivery to thehuman or animal body. Because the amphiphile has both polar and apolargroups which cluster to form polar and apolar regions, it caneffectively solubilise both polar and apolar compounds. In addition,many of the structures formed by amphiphiles/structuring agents in polarand/or apolar solvents have a very considerable area of polar/apolarboundary at which other amphiphilic compounds can be adsorbed andstabilised.

The formation of non-lamellar regions in the amphiphile/water,amphiphile/oil and amphiphile/oil/water phase diagrams is a well knownphenomenon. Such phases include liquid crystalline phases such as thecubic P, cubic D, cubic G and hexagonal phases, which are fluid at themolecular level but show significant long-range order, and the L₃“sponge” phase which comprises a multiply interconnectedthree-dimensional bi-continuous network of bilayer sheets which lack thelong-range order of the liquid crystalline phases. Depending upon theircurvature, these phases may be described as normal (mean curvaturetowards the apolar region) or reversed (mean curvature towards the polarregion). Where the spontaneous curvature of the lipid system is close tozero, the structures are typically lamellar, such as uni- ormulti-lamellar vesicles/liposomes and where the spontaneous curvature ismore negative or positive, micellar, cubic and hexagonal phasestypically dominate.

The non-lamellar (e.g. liquid crystalline and L₃) phases arethermodynamically stable systems. That is to say, they are not simply ameta-stable state that will separate and/or reform into layers, lamellarphases or the like, but are the thermodynamically stable form of themixture.

Both lamellar and non-lamellar systems have been investigated for theirproperties as carriers and/or excipients for dietary, cosmetic,nutritional, diagnostic and pharmaceutical agents but the non-lamellarsystems are thought to have considerable advantages in terms of theirhigh internal surface area between polar and apolar regions. This hasled to considerable investigation of non-lamellar phases particularly incontrolled-release formulations and for solubilising compounds ofrelatively low solubility.

As discussed above, a bulk non-lamellar phase is typically athermodynamically stable system. In addition, this bulk phase may bedispersed in a polar or non-polar solvent to form particles of anon-lamellar (especially liquid crystalline) phase in a bulk solvent.This allows the advantages of bulk non-lamellar phases to be applied insituations where use of a bulk non-miscible phase would cause problems,such as in parenteral applications. Further control of a compound'srelease profile may also be achieved by such a dispersion ofnon-lamellar particles.

Liquid crystalline or L₃ phase can be in or near thermodynamicequilibrium with the excess solvent and may be dispersed intocolloidally stable dispersions of non-lamellar particles. Such particlesmay be fully (i.e. thermodynamically) stable, or may gradually degrade,thereby providing control over the release profile for active agentsformulated therewith. The formation of dispersions can be spontaneous oras the result of mechanical force such as shearing or ultrasound. Thesenon-lamellar particles are of considerable interest in the delivery ofactive agents and have been proposed as carriers for many such actives.

A method for the formation of dispersed particles of non-lamellar phasein solvents such as water is described in U.S. Pat. No. 5,531,925. Suchparticles have a non-lamellar liquid crystalline or L₃ interior phaseand a lamellar or L₃ surface phase and may also contain activeingredients.

Known particles of liquid crystalline or L₃ interior phase may be formedby methods such as adding to this phase a solution of surface-phaseforming agent, stirring to form a coarse dispersion and fragmenting theresulting mixture.

In order to assess the presence of a liquid crystalline phase, theprospective liquid crystalline material may be examined by use ofsmall-angle X-ray diffraction (SAX), cryo-Transmission ElectronMicroscopy (cryo-TEM) or Nuclear Magnetic Resonance (NMR) spectroscopystudies. The sizes and size distributions of the dispersed particles maybe examined by light scattering, particularly by use of laser lightscattering instruments.

Dispersions containing active ingredients and particularly those forintravenous administration to the human or animal body are desirablycolloidal, that is they should be of a particle size no greater than 10μm, especially no greater than 5 μm and particularly no greater than 1μm. If particles within the dispersion exceed this size then thedispersion may not be colloidally stable and there is a considerablerisk of causing embolism when the preparation is administeredintravenously. Furthermore, it is desirable that the distribution ofparticle sizes be narrow to maximise control over the release of anyactive agent. Where a particulate composition is to be administered by amethod other than intravenously (e.g. orally, intramuscularly,subcutaneously, rectally or by inhalation), then the particles need notnecessarily be colloidal but it remains advantageous to provide a wellcharacterised and reproducible particle size distribution in order tocontrol the rate of decomposition of the particles and/or release of theactive agents.

The particle size of a particulate composition should also be stable tostorage over a considerable period of time. If the distribution ofparticle sizes changes significantly then the effective transport ratefor composition (e.g. due to diffusion and rate of release of any activeagent) may be adversely affected. Of still greater concern is thestability of particle sizes in a colloidal dispersion for intravenousadministration. If the particle size distribution of such a dispersionis not stable (e.g. to storage and distribution) then large particlesmay form over time and be dangerous when administered. Even if notdirectly dangerous, storage instability can cause significantvariability in pharmacokinetics, dynamics and/or efficacy.

In addition to control over particle size, it is desirable to maximisethe proportion of particles which are in the desired, non-lamellar,phase in order to maximise the beneficial effects of this in terms ofloading capacity, protective encapsulation, controlled release,reproducibility, etc. The proportion of lamellar particles such as uni-or multi-lamellar vesicles should therefore be minimised.

Known methods for the formation of dispersed particles of non-lamellarphase are highly effective, but typically produce a relatively broaddistribution of particle sizes and a considerable proportion of“contaminant” lamellar vesicular particles. Increasing the proportion offragmenting and/or stabilising agent (e.g. surfactant, copolymer and/orprotein) in the formulation or increasing the energy input of thehomogenisation process may be used to narrow the particle sizedistribution but at the expense of increasing the proportion of lamellarparticles.

One limitation of non-lamellar compositions presently available orsuggested is that they frequently rely upon lipids which are not welltolerated in vivo at elevated concentrations. In particular, commonlyused monoacyl glycerols (including the popular glyceryl monooleate—GMO)can be toxic if administered (especially parenterally) at highconcentrations, which can be dose-limiting. The possibility of toxicside effects from the lipid carrier can also limit the range ofindications for which an active agent is used to those of a highlyserous nature, where the risk of side-effects may be tolerated. Itwould, therefore, be a considerable advance to provide lipidcompositions which were formable and stable as particulate dispersions,showed predictable non-lamellar phase behaviour and had decreasedtoxicity, (e.g. as seen from haemolysis indices and/or acute toxicitystudies) when compared with widely used compositions (e.g. thoseincluding GMO). It would be of further advantage if such formulationswere formable and stable as colloidal sized particles (e.g. 0.05 toapproximately 2 μm diameter) and had a narrow, mono-modal, particle sizedistribution. It has been observed in the literature that stableparticular dispersions are particularly difficult to provide and thatonly lamellar dispersions are stable to storage for more than a few days(Kamo et al. Langmuir 19, 9191-9195 (2003)).

The present inventors have unexpectedly established that a mixture of atleast 3 amphiphilic components comprising a diacyl glycerol (DAG), atocopherol, or a diacyl phosphatidyl ethanolamine (PE) component, ormixtures thereof, a phosphatidyl choline (PC) component and a non-ionicstabilising component is highly effective in forming stable non-lamellardispersions and can show surprisingly low toxicity in vivo.

In a first aspect, the present invention therefore provides aparticulate composition comprising:

-   -   a) 5 to 90% of at least one phosphatidyl choline component,    -   b) 5 to 90% of at least one diacyl glycerol component, at least        one tocopherol, or mixtures thereof and    -   c) 1 to 40% (preferably 2-40%) of at least one non-ionic        stabilising amphiphile,        wherein all parts are by weight relative to the sum of the        weights of a+b+c and wherein the composition comprises particles        of at least one non-lamellar phase structure or forms particles        of at least one non-lamellar phase structure when contacted with        an aqueous fluid.

Preferred compositions of the present invention additionally contain atleast one active agent as described herein and may contain a solvent(particularly water or an aqueous solvent or solvent mixture). Thecompositions may also contain suitable carriers, excipients, fillers,stabilisers and similar components.

In a further aspect, the present invention provides a pharmaceuticalformulation comprising at least one composition of the invention and atleast one pharmaceutically tolerable carrier or excipient.

In a further aspect, the present invention provides a method for thetreatment of a human or animal subject comprising administration of acomposition of the present invention, optionally including an activeagent. In this aspect, the method of treatment is in particular a methodfor the treatment of inflammation and/or irritation, especially in abody cavity such as the gastrointestinal tract.

In a still further aspect, the present invention provides for the use ofa composition of the present invention in therapy, and in particularlyfor the use of a composition of the present invention, optionallyincluding an active agent, in the manufacture of a medicament for thetreatment of inflammation and/or irritation, especially in a body cavitysuch as the gastrointestinal tract.

The ternary amphiphilic compositions of the invention comprise at leastone PC component (component a), at least one DAG, at least onetocopherol, and/or at least one PE component (component b) and at leastone non-ionic stabilising amphiphilic component (component c). Componentc will, in particular facilitate fragmentation of the composition.

At least 5% by weight of total amphiphilic components (a+b+c) should becomponent a. Preferably this will be 5 to 50% and more preferably 10 to40%. Correspondingly, component b should be at least 5% by weight ofa+b+c, preferably 20 to 85%, more preferably 30 to 75%. Component cshould be present at 2 to 40%, preferably 3 to 35% and more preferably 5to 30% of the total weight of a+b+c.

In the ternary amphiphilic compositions, the phosphatidyl cholinecomponent “a” consists predominantly of lipids having the phosphatidylcholine polar group with two non-polar acyl chains attached by esterlinkages. This component is referred to herein, as in the literature, asphosphatidyl choline (PC) and may consist of one pure compound, such assynthetic dioleoyl phosphatidyl choline or, more preferably, will be amixture of PCs such as that derived from a purified natural source. PCis a particularly advantageous component in that it is widely andreadily available and can conveniently be purified from a variety ofnatural sources. The ability of the present invention to functioneffectively with naturally derived products, including mixed PCs, is aconsiderable advantage over certain other lipid components, such asdioleoyl phosphatidyl ethanolamine (DOPE) which are much more complex toextract & purify or must be synthesised, which makes them much moredifficult to produce and obtain on an industrial scale.

Typically, PCs extracted from natural sources will have a mixture ofacyl chains and this mixture will vary somewhat depending upon thetissue from which the extract was taken. Liver PC, for example has arelatively wide range of acyl chain lengths (at least C₁₆ to C₂₀) andhas significant proportions of acyl groups which are saturated and whichhave more than 2 unsaturations. In contrast, Soy PC typically containslargely C₁₆ to C₁₈ acyl groups with zero or two unsaturations. Thisallows the behaviour of the compositions of the present invention to besubtly altered by selecting an appropriate PC component or mixturesthereof. Preferred PCs include Egg, Heart, Brain, Liver and particularlySoy PC (SPC). The PC or any portion thereof may also be hydrogenatedwhere a higher proportion of saturated PCs is desired.

Since the PC component of the present invention is preferably a naturalextract, it is common for a small amount of non-PC “contaminant” to bepresent. The exact level of purity of the PC component in terms ofcontent of lipids having other polar groups will depend upon theparticular application for which the compositions of the invention areto be used. The important factor is that the phase behaviour, remarkablyhigh stability and remarkably low toxicity of the compositions should bemaintained by choice of suitably pure components. So long as thisremains acceptable then the purity of the PC will be of relatively minorsignificance. As a general guide, however, the PC component willcommonly contain no more than 10% by weight of lipids with non-PC polargroups. In some embodiments this will preferably be no more than 5% andmore preferably no more than 2% by weight.

In the compositions of the invention, component b) is a diacyl glycerol(DAG), and/or a tocopherol. These may consist of a single, pure diacylglycerol, or tocopherol, may be a mixture of diacyl glycerols, and/ortocopherols, or may be a purified natural extract with a high diacylglycerol, and/or tocopherol content. Preferred diacyl glycerols haveacyl groups independently having 10 to 24 carbons, preferably 12 to 20carbons, and most preferably 14 to 18 carbons. Saturated and/orunsaturated acyl groups are suitable but groups with one, two or threedouble bonds are preferred. The acyl groups may be the same ordifferent. A highly preferred DAG is glycerol dioleate (GDO) andmixtures thereof.

As used herein, the term “a tocopherol” is used to indicate thenon-ionic lipid tocopherol, often known as vitamin E, and/or anysuitable salts and/or analogues thereof. Suitable analogues will bethose providing the phase-behaviour, stability and lack of toxicitywhich characterises the compositions of the present invention and willgenerally not form liquid crystalline phase structures as a purecompound in water. The most preferred of the tocopherols is tocopherolitself, having the structure below. Evidently, particularly where thisis purified from a natural source, there may be a small proportion ofnon-tocopherol “contaminant” but this will not be sufficient to alterthe advantageous phase-behaviour, stability and lack of toxicity.Typically, a tocopherol will contain no more than 10% ofnon-tocopherol-analogue compounds, preferably no more than 5% and mostpreferably no more than 2% by weight.

As with the PC component, component b, for example diacylglycerol,and/or tocopherol, may be provided as a natural extract. This hassignificant advantages in terms of availability and reliability of thematerials. Where a DAG component is a natural extract, however, it islikely that a small amount of non-DAG lipid will remain present. As withthe PC component, the crucial test of purity for the DAG component willbe that the composition provides the advantageous stability andnon-toxicity of the present invention. Typically the DAG component willhave no more than 15% by weight of other lipids, preferably no more than10% and more preferably no more than 5%. DAGs (as described herein) arepreferred component b)s.

A preferred combination of constituents for component b) is a mixture ofat least one DAG (e.g. GDO) with at least one tocopherol. Such mixturesinclude 2:98 to 98:2 by weight tocopherol:GDO, e.g. 10:90 to 90:10tocopherol:GDO and especially 20:80 to 80:20 of these compounds.

A highly preferred combination of components a and b is PC with DAG,wherein both components have at least 50% C18:1 (oleoyl) and/or 50%C18:2 (linoleoyl) acyl groups. Soy PC and Egg PC are particularlypreferred Examples. A preferred a weight ratio for these is between 1:5and 3:2, most preferably 2:5 to 4:5 PC:GDO. One measure of thebiological activity of a lipid is its solubility in water or aqueoussolutions. Components with relatively high aqueous solubilities maintaina higher equilibrium concentration of dissolved lipid monomer insolution and this can be at least partially responsible for the observedbiological effects. The commonly used “glycerol monooleate” (GMO), forexample, has an equilibrium water solubility of the order of 10⁻⁷ M atroom temperature and greater at physiological temperature. In contrast,preferred diacyl glycerols and diacyl phosphatidyl ethanolamines mayhave a solubility of no more than 10⁻⁸ or more typically 10⁻⁹ M at roomtemperature, preferably 5×10⁻¹⁰ M and more preferably 10⁻¹⁰ M or less.The minimum desirable solubility is generally around 10⁻¹⁵ M. Inparticular, at high dilution, the stability of the non-lamellar systemwill depend upon the rate at which lipid molecules leave the surface ofthe structured material and diffuse into solution. The stability of adispersion of non-lamellar particles will thus be directly related tothe solubility of the monomer in the solvent.

Component c acts as a fragmentation agent and helps both in the controland stability of particle phase behaviour and in encouraging andstabilising the fragmentation of the non-lamellar phase into particles.Component c will be present at a level sufficient to bring about thefragmentation of the composition and/or to stabilise the fragmentednon-lamellar phase particles. Such fragmentation may be spontaneous ormay require physical fragmentation such as by shearing and/orultrasonication. The skilled worker will have no difficulty in assessingwhether any composition contains sufficient fragmentation agent in viewof the Examples herein.

The non-ionic stabilising amphiphile “c” is, in general, a componentwhich improves the stability of the dispersion, particularly ascolloidal particles. The preferred form of these non-ionic amphiphilesis a non-ionic lipid grafted with a polyoxyethylene and/orpolyoxypropylene chain (or a copolymer thereof).

Such compounds are, for example, polyoxyalkyl grafted fatty acids, orsubstituted fatty acids (especially hydroxylated fatty acids),polyoxylalky grafted lipids or polyols having polyoxyalkyl groupsgrafted to one or more (preferably all) alcohol moieties and having oneor more fatty acid chains joined to the opposite end of one or more ofthe polyoxyalkyl chain. Examples include polyethylene glycol (PEG)sterate, PEG disterate, PEG laurate, PEG oleate, polyethoxylated casteroil, PEG-DOPE, PEG-(4-hydroxy sterate) (Solutol),PEG-sorbitan-monolaurate (Polysorbate 20 or 21),PEG-sorbitan-monopalmitateate (Polysorbate 40), PEG-sorbitan-monosterate(Polysorbate 60 or 61), PEG-sorbitan-tristerate (Polysorbate 65),PEG-sorbitan-monooleate (Polysorbate 80 or 81), PEG-sorbitan-trioleate(Polysorbate 85), PEG-sorbitan-monoisooleate (Polysorbate 120) andd-alpha tocopheryl polyethylene glycol 1000 succinate (Vitamin E TPGS).PEG chains may be attached to the other components of the amphiphile byester or ether bonds as appropriate. Typically, the totalpolyoxyalkylene content of a molecule of component c will be no morethan 50 monomers, preferably no more than 30 monomers. Most preferredare Polysorbates 20 and 80, solutol, d-alpha tocopheryl polyethyleneglycol 1000 succinate (Vitamin E TPGS) and polyethoxylated caster oil.

The present inventors have now established that the stability of thenon-lamellar particulate dispersions shows considerable dependence onthe type of stabilising agent used. In particular, the dispersing agentsindicated above are highly preferred because it has been found that highmolecular weight surfactants are notably less stabilising to theparticulate dispersion than the lower molecular weight compoundsindicated. In one embodiment, therefore, component c) should comprise orpreferably consist essentially of, or consist of surfactants with amolecular weight below 10,000 amu, preferably below 8,000 and morepreferably below 5000 amu. Similarly, component c) should preferably notcontain a block-copolymer surfactant, especially one with molecularweight above the ranges indicated above. This is particularly surprisingbecause higher molecular weight surfactants have been shown as effectivefor stabilising related compositions in lamellar form.

One important aspect of the present invention is that the compositionsmay be formulated as preconcentrates, containing a cosolvent, asdiscussed herein. These preconcentrates are particularly suitable foruse as controlled release system in parenteral applications. In thisuse, the most useful ratios between c/a+b+c are 1-30% of component c,more preferably 3-25%. For compositions forming depots intended forrelease over around one week, the most preferred range is 3-10% c.

The amphiphile components of the compositions of the invention mayconsist essentially of, or consist of, components a), b) and c) only(plus any active if this is amphiphilic). In this embodiment at least95%, preferably at least 98% and most preferably substantially 100% ofthe amphiphile components will be one of these. Alternatively, anadditional, optional, amphiphilic component d) may be present in amountsup to 10% by weight of a)+b)+c)+d), preferably up to 8%, more preferablyup to 5%. This component d) may be any suitable amphiphile, such as anatural or synthetic lipid or a derivative or analogue thereof. A highlypreferred component d) is an ionic lipid, such as a fatty acid orbiotolerable salt thereof.

The compositions of the present invention comprise non-lamellarparticles or are compositions which form such particles on contact withan aqueous fluid. Such a fluid may be a fluid for delivery to a subject(e.g. water or sterile saline) or may be a body fluid, particularlygastric fluid, intestinal fluid, fluid at mucosal surfaces, blood orintercellular fluid.

As use herein, the term “non-lamellar” is used to indicate a cubic,hexagonal, L₂ or L₃ phase structure or any combination thereof, asopposed to lamellar structures as found in lamellar phase orliposomes/vesicles. Where a particle is described as having anon-lamellar phase or structure, this indicates that at least theparticle interior has this structure. Many of the particles will havetwo distinct regions, an internal region and a surrounding surfaceregion. The surface region, even in a “non-lamellar” particle may belamellar or crystalline and may be any phase including highly orderedcrystalline layers, liquid crystal phases and virtually orderless fluidlayers.

The term “lamellar particles” is used herein to indicate vesicularparticles (e.g. liposomes) characterised in that they comprise one ormore outer lamellar bilayers of amphiphile, surrounding an inner solventcompartment.

In one aspect of the present invention, the compositions comprisenon-lamellar particles. This indicates that of the (preferablycolloidal) particles present, at least 50%, preferably at least 75% andmost preferably at least 85% (as measured by volume) are non-lamellar(e.g. as judged by laser diffraction combined with cryo-TEM or SAXS). Inan alternative aspect of the present invention, the compositions formnon-lamellar particles on contact with an aqueous fluid. This indicatesthat upon contact with an aqueous fluid (as described herein) at least50%, preferably at least 75% and most preferably at least 85% of theparticles (as measured by volume) become non-lamellar particles.

In a preferred embodiment of the present invention, the presentcompositions comprise or generate particles of reversed hexagonal and/orL₃ phase. Most preferably the compositions comprise or generateparticles of L₃ phase. L₃, otherwise known as “sponge” phase lacks thelong-range order of a true-liquid crystalline phase but consists ofmultiply interconnected sheets of lipid bilayer where these“interconnections” do not adopt the regular arrangement seen in cubicliquid crystalline structures.

In an alternative an also highly advantageous embodiment, thecompositions of the invention may form I₂ or L₂ non-lamellar phases. TheI₂ phase is a reversed cubic liquid crystalline phase havingdiscontinuous aqueous regions. This phase is of particular advantage inthe controlled release of active agents and especially in combinationwith polar active agents, such as water soluble actives. The L₂ phasehas similar advantages and in a so-called “reversed micellar” phasehaving a continuous hydrophobic region surrounding discrete polar cores.

For many combinations of lipids, only certain non-lamellar phases exist,or exist in any stable state. It is a surprising feature of the presentinvention that compositions as described herein frequently exhibitnon-lamellar phases which are not present with many other combinationsof components. In one particularly advantageous embodiment, therefore,the present invention relates to compositions having a combination ofcomponents for which and I₂ and/or L₂ phase region exists when dilutedwith aqueous solvent. The presence or absence of such regions can betested easily for any particular combination by simple dilution of thecomposition with aqueous solvent and study of the resulting phasestructures by the methods described herein.

Where an active agent is formulated in a composition of the invention,the active agent will frequently have an effect upon the phase behaviourof the structuring agent(s). For example, certain active agents (such ascyclosporin A) introduce greater negative curvature than somestructuring agents and at high concentrations may cause the formation ofhighly negatively curved phases, such as the reversed micellar L₂ phaserather than a cubic or hexagonal liquid crystalline phase. Nonetheless,such an active agent could be formulated into, for example, a reversedhexagonal phase by formulation with a mixture of components a, b and chaving a less negative spontaneous curvature. By this method, theoverall mixture provides the appropriate negative curvature to allow usein the compositions of the invention.

The skilled worker will be able to use standard methods to assess thedegree of spontaneous curvature of any particular structuring agent (ormixture thereof with other components) or the effect on this byincluding an active agent. This might be done, for example, by studiesof the bulk phase behaviour of each structuring agent in water andsubsequent studies with varying concentrations of active agent included.The phases can be examined by any of the methods indicated herein (e.g.polarised light, SAXS, cryo-TEM etc.) and an appropriate blend ofcomponents chosen for each case. In some circumstances, where the effectof the active agent on the phase behaviour of the mixture issignificant, the structuring agent(s) chosen may not provide the desirednon-lamellar phase in themselves (e.g. may have too small or too greatspontaneous curvature) but will generate this phase only when alsoformulated with the active agent. The equilibrium phase may thus changefrom, for example, cubic to hexagonal liquid crystalline phase uponaddition of the active agent.

In one preferred aspect, the compositions of the present inventioncomprise at least one active agent. Suitable active agents include humanand veterinary drugs and vaccines, diagnostic agents, “alternative”active agents such as plant essential oils, extracts or aromas, cosmeticagents, nutrients, dietary supplements etc. Examples of suitable drugsinclude antibacterial agents such as β-lactams or macrocyclic peptideantibiotics, anti fungal agents such as polyene macrolides (e.g.amphotericin B) or azole antifungals, anticancer and/or anti viral drugssuch as nucleoside analogues, paclitaxel, and derivatives thereof, antiinflammatories, such as non-steroidal anti inflammatory drugs,cardiovascular drugs including cholesterol lowering and blood-pressurelowing agents, analgesics, anaesthetics, antidepressants includingserotonin uptake inhibitors, vaccines and bone modulators. Diagnosticagents include radionuclide labelled compounds and contrast agentsincluding X-ray, ultrasound and MRI contrast enhancing agents. Nutrientsinclude vitamins, coenzymes, dietary supplements etc. The active agentsfor use in the present invention will generally not be any of componentsa, b, or c as described herein. Other preferred active agents includeinsulin and insulin analogues, growth hormones such as human growthhormone (hgh) immunosuppressants such as tacrolimus and cyclosporine A,peptide drugs such as those described herein, including octreotide,salmon calcitonin, desmopressin, somatostatin, antibodies and antibodyfragments, nucleic acids including antisense and interfereing nucleicacids (e.g. siRNAs) and vaccines.

In one preferred aspect of the present invention, the composition of theinvention is such that an I₂ or L₂ phase, or a mixture thereof is formedupon exposure to aqueous fluids and a polar active agent is included inthe composition. Particularly suitable polar active agents includepeptide and protein actives, including those listed below. Of particularinterest in this aspect are the peptides octreotide and othersomatostatin related peptides, the polar active chlorhexidine (e.g.chlorhexidine digluconate or chlorhexidine dihydrochloride) andbisphosphonates (e.g. ibandronate, zoledronate, alendronate,pamidronate, tiludronate etc.).

One particularly suitable class of active agents for inclusion in anyappropriate aspect of the invention are the peptide/protein actives.These include; hormones & hormone derivatives such as somatotropin,somatostatin (& analogues), calcitonin (human or salmon), oxytocin,gonadorelin (and derivatives such as leuprolide; goserelin andtriptorelin), vassopresin, (and derivatives such as desmopressin andfelypressin), follitropin-alpha and -beta, human chorionicgonadotropin-beta, thyrotropin alpha, secretin (e.g. porcine),bradykinin, hypotensive tissue hormone, insulin α and insulin β;antiviral, antibacterial and antifungal peptides including,interferon-alpha 1/13, interferon-alpha 2, interferon-beta,interferon-gamma, (including recombinant forms), tachyplesin i, tuftsin,magainin i and ii, indolicidin (e.g. bovine), protegrin (e.g. swine),polyphemusin i & ii, polymixin b, gramicidin s; interleukins (ils)including il-1 alpha, hematopoietin-1, il-1 beta, catabolin, il-2,t-cell growth factor (tcgf) (aldesleukin), il-3, haematopoietic growthfactor, il-4, b-cell stimulatory factor, il-5, t-cell replacing factor,il-6, b-cell stimulatory factor, il-7, il-8, neutrophil-activating,il-9, t-cell growth factor p40, il-10, cytokine synthesis inhibitoryfactor, il-11, adipogenesis inhibitory factor, il-13, il-15, il-17,cytotoxic t lymphocyte-associated antigen 8, il-18, interferon-gammainducing factor, il-19, melanoma differentiation associated protein-likeprotein, il-20, four alpha helix cytokine zcyto 10, il-24, melanomadifferentiation associated protein 7, il-26; and other peptides &proteins, including intercellular adhesion molecule 1, pneumadin,alteplase, interleukin-1 receptor antagonist, gmcsf, filgrastim (g-csf),lepirudin, becaplermin, ospa, avicine, tubulysins a-f, contakulin g(cgx-160), alpha conotoxin-like peptides, (see wo 02/079236), andmellitin.

In the methods of treatment of the present invention, as well as in thecorresponding use in therapy and the manufacture of medicaments, anactive agent is not always necessary. In particular, lipids,particularly phospholipids such as PC have been implicated as highlybeneficial in themselves for the treatment of certain conditions(including those described herein below). Without being bound by theory,it is believed that suitable lipids, such as those in the formulationsof the present invention, form protective layers over and around manystructures of the body, such as the linings of many body cavities andthe contact surfaces of joints. These layers may serve as protectionfrom adhesion and attack by a wide variety of chemical and biologicalagents (such as on gastric surfaces and in the lining of the GI tract),may act as lubricants (particularly in joints but crucially also on thelinings and membranes surrounding many internal structures such as heartand lungs), and may additionally contribute to cell wall repair byallowing lipid exchange and dilution of undesirable membrane-bound andmembrane-soluble agents. The lipid nature of the compositions also formsa harmless substrate for unwanted inflammatory lipase enzymes such asphospholipases such as phospholipase A₂ (PLA₂).

In an alternative embodiment of the methods of treatment andcorresponding uses of the present invention, suitable actives may beincluded, either as the sole beneficial agent, or to complement theeffect of suitable lipid components. Suitable actives will typically besuitable for the treatment of inflammation and/or irritation, such assteroidal and non-steroidal anti-inflammatory drugs and local immunemodulators. Examples of such agents are well known and includecorticosteroids such as prednisone methylprednisolone andhydrocortisone, and derivatives of nonsteroidal anti-inflammatorycompounds such as benzydamine, paracetamol, ibuprofen and salicylic acidderivatives including acetyl salicylate and 5-amino salicylates. Localinhibitors of inflammatory pathways are also suitable, including theantigen recognition suppressors methotrexate, azathioprine or6-mercaptopurine and phospholipase inhibitors, such as PLA₂ inhibitors.In this context it is noteworthy that the composition of the inventionis suitable for intra-articular administration into the synovial fluidwhere phospholipids in addition to being controlled release carriershave known beneficiary effects relating to joint lubrication.

Suitable loadings for the active agents will be established by referenceto their known doses, bearing in mind the route of administration andthat the compositions of the invention may provide a greater biologicaluptake of active agent than known formulations.

One particularly advantageous aspect of the compositions of theinvention is that a very high level of active agent can be incorporated.In particular, compositions containing a proportion of water or acosolvent (as described herein), are highly effective in solubilisinghigh levels of active agents of many types. The compositions may thuscontain at least 2% of an active agent, preferably at least 5% and morepreferably at least 10% of an active agent. Advantageously, up to 20% byweight of active agent may be incorporated.

The amphiphile based particles of the invention (including those formedor formable from the compositions of the invention) may desirably alsobe modified with surface active agent(s) (especially a polymer) e.g. astarch or starch derivative, a copolymer containing alkylene oxideresidues (such as ethylene oxide/propylene oxide block copolymers),cellulose derivatives (e.g. hydroxypropylmethylcellulose,hydroxyethylcellulose, ethylhydroxyethylcellulose,carboxymethylcellulose, etc) or graft hydrophobically modifiedderivatives thereof, acacia gum, hydrophobically modified polyacrylicacids or polyacrylates, etc. The surface active polymer may also be usedto provide a functional effect on the surface of the particles, forexample, in order to selectively bind or target the particles to theirdesired site of action. In particular, polymers such as polyacrylicacids, hyaluronic acids, gellan gum or chitosans may be used to providemucus adhesive particles. Such particles will thus tend to remainlocalised, thus increasing the spatial control over the active agentrelease. Compositions of the invention comprising such surface modifiedparticles form a further embodiment of the invention.

In colloidal compositions, the average particle size will typically bein the range 0.1 to 0.6 μm, for example as determined by lightscattering methods (e.g. laser diffraction). Preferably, no more than 1%of particles will be outside the range 0.05 to 1.5 μm, more preferably,not more than 0.1% will be outside this range, and most preferably nodetectable (by laser diffraction) proportion of particles will beoutside this range. In non-colloidal formulations the average particlesize will typically be in the range 10 to 200 μm.

A highly significant advantage of the present invention is, furthermore,that the colloidal formulations are typically physically stable tostorage over extended periods at ambient temperature. Such formulationsshould be essentially stable in terms of phase behaviour, particle sizeand particle size distribution for periods of at least 10 days at roomtemperature, more typically at least 3 months, preferably at least 6months and more preferably 12 months or more. In contrast, knowndispersions of similar particle size may have particle sizes stable forless than 10 days at room temperature (see e.g. Kamo et al supra) Thisis a particular advantage of compositions of the present inventioncomprising components a+b+c, since compositions of components a+b in theabsence of component c are typically less stable to storage.

A particle size distribution can be considered essentially stable tostorage if the mean particle size increases no more than two fold duringthe storage period. Preferably, the mean size should increase no morethan 50% and more preferably no more than 20% during the storage period.Similarly, the width of the distribution at half-height shouldpreferably increase by no more than 50%, more preferably by no more than20% and most preferably no more than 10% during the storage period.Where a distribution is monomodal, it should preferably remain monomodalduring the storage period. In a highly preferred embodiment, theparticle size distribution of the compositions of the invention altersin mean particle size and particle size distribution width athalf-height by no more than 10% and remains monomodal on storage for theperiods indicated above.

It is particularly important in the case of colloidal dispersions foruse in intravenous or intra-arterial administration that the particlesize distribution be stable during storage and use. A compositioncontaining even a relatively small component of non-colloidal particlesmay cause embolism, or at least unpredictable rates of release uponadministration directly to the blood stream. Similarly, the controlledrelease of an active agent may be dependent upon a reliable particlesize distribution in a composition for administration by any otherroute. Pharmaceutical, diagnostic and veterinary products are alsodesirably stable to storage for several months or the cost andavailability of the product is significantly adversely affected.

In an additional important and highly preferred embodiment of theinvention, liquid compositions of the invention may be prepared assolvent mixtures. Such liquid precursors will comprise components a, b,c, a cosolvent and optionally an active agent. The liquid precursorscontaining an active agent can, for example, be filled in capsules andnon-lamellar particles form when contacted with GI-fluid. Similarly, aliquid precursor may be provided in an ampoule for dispersion in a fluid(e.g. isotonic saline) prior to injection or may be injected directlyand form non-lamellar particles in vivo upon contact with a body fluid.Most importantly the present inventors have unexpectedly discovered thatby varying the amount of component c it is possible to tune the in vivorelease duration in the time window from a few hours up to severalweeks. Furthermore, high initial drug concentrations can be avoided thusreducing potential local and systemic side effects.

Co-solvents should generally be miscible or at least partially solublewith water and should be tolerable in the application in which thecomposition will be used. Organic solvents having 1 to 6 carbon atomsand preferably at least one oxygen substituent and water-solublepolymers thereof are preferred. Suitable classes of co-solvents arealcohols (including polyols), ketones, esters, ethers, and polymersthereof. Typical co-solvents are ethanol, isopropanol,N-methyl-2-pyrrolidone (NMP), propylene glycol, PEG400 and glycerol.Ethanol is particularly suitable. The solvent may be added at a level upto about 10 to 20% (by weight) of total lipid.

The compositions of the present invention may be formed by preparing adispersion of components a, b, and c in a solvent (such as an aqueoussolvent) and then optionally treating the dispersion with one or morecycles of heating and cooling.

Dispersions of particles comprising components a, b and c are formed aspre-formulations prior to the optional heat treatment cycles. Thispre-formulation may be prepared by established methods, such as thoseindicated in the present Examples and in U.S. Pat. No. 5,531,925, WO02/02716, WO 02/068561, WO 02/066014 and WO 02/068562 and may itself bea composition of the invention. The disclosures of these and allreferences cited herein are hereby incorporated herein by reference.Such methods include:

i) Adding an amphiphile/water liquid crystal phase (such as component ain water) to an aqueous solution of fragmentation agent (such ascomponents b and/or c) and either allowing natural fragmentation of themixture or accelerating the process with, for example, mechanicalagitation, vortexing, roto-stator mixing, high-pressure homogenization,microfluidisation and/or ultrasound; orii) Adding a mixture of a+b+c (optionally containing at least onebioactive agent) to a solvent (e.g. aqueous solution) and agitatingdirectly.

A further method by which dispersion containing active agents may beprepared, particularly from liquid crystalline phases, is by dissolutionin super-critical carbon dioxide (sc-CO₂) or an alternative processingsolvent, such as light alcohols (e.g. methanol or ethanol), suitable fordissolving and lowering the viscosity of the composition. In particular,liquid crystalline phase, such as bulk cubic or hexagonal phase, isoften highly viscous and can be difficult to handle and mix.Consequently, if the liquid crystalline phase is to be prepared as abulk liquid and subsequently loaded with active agent, the mixingrequired to provide even distribution of the active agent is difficultto achieve. In the super-critical region of the pressure/temperaturediagram (typically at room temperature or above and at 150 bar orgreater), carbon dioxide forms a highly effective solvent and may beused to reduce the viscosity of the liquid crystalline phase and promoteeffective mixing and loading with active agents. The sc-CO₂ may then beremoved (e.g. by reducing the pressure) and the loaded bulk phasedispersed in solvent, as discussed above. The use of sc-CO₂ in formationof active-agent loaded dispersed liquid crystalline phases (especiallythose of the present invention) thus forms a further aspect of theinvention.

The phase behaviour and size distribution of particulate formulations ofthe invention may be controlled by one or more (preferably one) cyclesof heating and cooling. Such cycles can be used to convert lamellarparticles to non-lamellar form, and/or to reduce the spread of particlesizes. The stability of the particles may also be improved by thismethod.

A heat cycle brings the composition, with or without the active agentpresent, up to a temperature sufficient to provide conversion of atleast a portion of the particles to non-lamellar phase upon cooling toambient temperature. This will typically involve heating to around90-150° C. for 1-30 min followed by cooling to ambient temperature. Moretypically a heat cycle will involve heating to 100-120° C. for 2-20minutes before cooling. The most suitable conditions will vary in detailbetween compositions but will be readily established by the skilledworker.

In the heat cycling process, the mean particle size typically increasesslightly but the particle size distribution is reduced.

In a further aspect, the present invention thus also provides a methodfor the formation of non-lamellar particles comprising forming a mixturecomprising;

-   a) 5 to 90% of at least one phosphatidyl choline component,-   b) 5 to 90% of at least one diacyl glycerol component, at least one    tocopherol, or mixtures thereof, and-   c) 2 to 40% of at least one non-ionic stabilising amphiphile,    wherein all parts are by weight relative to the sum of the weights    of a+b+c and dispersing said mixture in an aqueous fluid. The method    preferably also comprises at least one heating and cooling cycle as    described herein and the aqueous fluid may be water, an aqueous    solution suitable for injection, a body fluid or any other suitable    fluid as indicated herein. The mixture may consist purely of the    amphiphiles a-c or may also contain other components such as active    agents and/or water miscible solvents, as illustrated in the    Examples below. The method is also optionally followed by a drying    step (such as spray drying or freeze drying) whereby to form the    compositions into the form of a powder.

The presence of particles in non-lamellar form will preferably beassessed from a set of cryo-transmission electron microscopy particleimages, preferably showing a sample of more than 20, preferably morethan 30 and most preferably at least 50 particles. The presence ofnon-lamellar particles may also be assessed by X-ray scatteringexperiments.

Since the heat treatment method can be used to convert lamellarparticles to non-lamellar form, it is not essential that thepre-formulation particles be non-lamellar. Thus, any of the well-knownmethods for formulating lipids into vesicles may be used to createpre-formulations for use in heat treatment methods of the presentinvention. Suitable methods include, for example, sonication orextrusion (such as through a polycarbonate membrane). Such methods willbe well known to those of skill in the appropriate art.

The pre-formulations should, preferably, be formulated such that thethermodynamically stable state at ambient temperature is non-lamellarthis will generally be the case due to the specific choice of componentsa, b and c, and the proportions thereof. Alternatively, the non-lamellarform may be a thermodynamically meta-stable state. Where present, theactive agent may be incorporated into the particles prior to and/orafter heat cycling. Where more than one heat cycle is used, the activeagent may be incorporated between cycles. Where the active agent is heatsensitive (e.g. peptide or protein) the active agent is preferablyincorporated after heat cycling is complete. In contrast, where theactive agent is stable to the heat cycling method, this method (heatcycling in the presence of the active agent) can be used to provide veryhigh loading levels of active agent, which remain stable for longperiods.

The particles (which may have been heat treated or may be subsequentlyheat treated) may be concentrated (e.g. by ultrafiltration or dialysis)and/or dried, for example by spray drying, fluid bed drying or freezedrying. In the case of dried particles, the drying process may befollowed by particle size enlargement through single or repeatedagglomeration and granulation steps. The concentrated, dried and/oragglomerated particle formulations thus formed may be used as such orhydrated and/or dispersed to yield non-lamellar particle dispersionssuitable for use in the delivery of active substances, especially invivo. Such concentrated, dried and/or agglomerated particle formulationsand the dispersions resulting from their re-suspension/hydration form afurther aspect of the present invention.

The dry (by which is meant functionally dry rather than being completelydevoid of solvent) powder compositions of the invention may beresuspended to give colloidal or non-colloidal dispersions in a suitable(especially aqueous) fluid. Alternatively, the dry compositions may bedissolved in a suitable co-solvent, as described herein, andadministered whereby to form non-lamellar structures in vivo uponcontact with a body fluid. These aspects of the invention areparticularly suited for intramuscular and/or subcutaneous injection andmay form a long-lasting non-lamellar structure from which active agentmay be slowly released over a period of days or weeks. Such slow-releaseformulations may be generated from any suitable composition of theinvention but are particularly suited to generation from re-suspendedpowders.

Semi-solid (e.g. gel, waxy solid) compositions may be prepared by use ofa polymeric agent in the compositions of the invention. Such semi-solidprecursors will comprise compositions of the invention as describedherein and additionally at least one polymeric solidifying agent.Typically, such compositions comprise components a, b, c, a polymericagent, optionally a co-solvent and optionally an active agent. Thesemi-solid precursors are typically liquefiable by heat and can, forexample, be filled in capsules, moulded etc. The semi-solid compositionsof the invention may be resuspended to give colloidal or non-colloidalnon-lamellar particle dispersions in a suitable (especially aqueous)fluid. Alternatively, non-lamellar structures form when contacted withaqueous body fluids e.g. GI-fluid. The polymeric solidifying agent is apreferably biotollerable polymer, preferably having a melting pointbetween 35 and 100° C., more preferably 40-95° C. and most preferably45-90° C. A particularly preferred polymeric agent is polyethyleneglycol (PEG) with molar mass in the range of 950-35000, most preferably1000 to 10,000. PEG 4000 is a highly preferred example.

The formulations of the present invention comprise at least onecomposition of the invention and at least one suitable carrier orexcipient. Where the formulation is a pharmaceutical formulation, thecarriers or excipients will be pharmaceutically tolerable.

The compositions may be formulated with conventional pharmaceuticalcarriers, diluents and/or excipients such as aqueous carriers (e.g.water for injections), binders, fillers, stabilizers, osmolalityadjusting agents, antioxidants, effervescing agents, pH buffers andmodifiers, viscosity modifiers, sweeteners, lubricants, emulsifiers,flavours, coating agents (e.g. gastric juice resistant coatings) etc.

Formulations comprising a composition of the invention and at least onepharmaceutically acceptable carrier and/or diluent may be formulated inany known dosage form including as suspensions in liquid, powders,tablets, capsules, coated capsules, coated tablets, aerosols,suppositories, drops, creams, transdermal patches, sprays etc. Where thecomposition of the invention has been dried, this may be formulated in asuitable form (such as a powder) for resuspension in an appropriatemedium (such as purified water or a solution of physiologicalosmolality) prior to administration. The formulations and pharmaceuticalcompositions may be administered by any suitable method includingorally, ophthalmicly, by inhalation, parenterally (e.g. byintramuscular, subcutaneous or intravenous injection or infusion),topically, rectally etc. Parenteral compositions are preferred since thepresent invention provides a remarkable combination of high stability(in particle size and phase structure) and very low parenteral toxicity.Topical compositions are, however, also highly effective and pump-sprayor pressurised spray dispersions may be used for dermal, nasal, andintra-oral (especially buccal) applications. Rectal administration as aconcentrated dispersion or solidified suppository are also highlysuitable.

The formulations, compositions and methods of the invention relating tothe treatment of inflammation or irritation, are particularly suitablefor addressing inflammation and/or irritation in a body cavity.Administration to a body cavity is thus highly suitable in this aspectand will be carried out by a method suitable for the cavity beingtreated. Mouthwashes, for example, may be suitable for oral or buccalcavities, while other parts of the GI tract may be suitably treated byoral formulations, including dispersions and dry pre-formulations, andrectal formulations such as enemas or suppositories. Rinses andpesseries are similarly suitable for vaginal delivery.

The compositions of the present invention are highly suitable fortreating inflammation in a body cavity because of the highly bioadhesivemature of the non-lamellar phase and the resulting long-lasing effects.The ability of the formulations to comprise or disperse intonon-lamellar particles, which are then easily transported anddistributed around the site of application is also of importance, aswell as the inherently soothing and highly biocompatible nature of theconstituents.

The methods of treatment and corresponding uses of the present inventionare thus most applicable to inflammatory diseases and inflammationcaused by, for example, wounding or abrasion. Especially suitable areinflammatory diseases affecting at least one body cavity. Diseases ofthe GI tract are highly suitable for treatment with the compositions ofthe present invention, particularly inflammatory bowel disease includingCrohn's disease and ulcerative collitus. Similarly, application to abody cavity during surgery may also be used to take advantage of theproperties of the formulations. They may thus be directly applied, forexample by spraying or painting, to sooth inflammation resulting from orexposed during surgery and also to reduce the tendency of surgicallymanipulated tissue to “stick” and/or form adhesions/bridges at unwantedsites.

A particular advantage of the compositions of the present invention istheir remarkably low toxicity, particularly when administeredparenterally. In particular, the present compositions can show aremarkably low acute toxicity when administered by intravenousinjection. In a preferred aspect, the present invention thus providescompositions of the invention showing no acute toxicity by intravenousinjection in rats to a level of at least 200 mg/kg body weight,preferably at least 600 mg/kg and more preferably at least 1 g/kg bodyweight.

As mentioned a further key advantage of the compositions of the presentinvention is that they can be used to generate “short term” depotcompositions. In particularly, immediate release formulations of activeagents are common and coatings etc can sometimes be used to provideformulations that will release active agents over a period up to around12 hours. In contrast, long-acting “depot” injections typically comprisesolutions or suspensions of polymers, such as poly-lactate-co-glycolate,or implanted physical pumps, which are driven by osmotic pressure. Thesemethods typically provide release over a period of a month or more andtypically require complex preparation and/or administration. Incontrast, there are few methods for releasing active agents over a 1-30day period, especially over a 1-14 day period, and most preferably overa 2-7 day period, as might be required, for example, for post-operativeanalgesia or a course of antibiotics. The compositions of the inventionhave one of more of the following advantageous properties provided as aresult of their specific composition:

They provide ready-to-inject compositions with little or no preparationrequired;

They avoid the lengthy preparation and administration typically requiredfor depot products;

They can be injected directly from a pre-filled injection devicecontaining the composition (which also forms an aspect of theinvention);

They are stable to storage, as indicated above;

They can be injected through a fine bore needle (e.g. less than 20gauge, preferably 23 gauge or less, more preferably 27 gauge orsmaller);

They can be injected effectively intramuscularly or subcutaneously orintracavitary;

High levels of active agent can be incorporated (as indicated herein);

A specifically favoured short-duration depot is one comprising acomposition of the present invention with GLP-1 or an analogue orderivative thereof (preferably at a dose of 0.1 to 20 mg) forsubcutaneous or intramuscular injection. Such a depot could providesustained release of the GLP-1 analogue over 2-14 days, preferably 5 to10 days. The most suitable use for such a composition would be in thetreatment of diabetes (especially type II) or in the manufacture of amedicament for such a use.

Glucagon-like peptide (GLP)-1 is a potent glucoregulatory hormone thatis released from intestinal L cells into the circulation in response tonutrient ingestion and neural and endocrine stimuli. Structurally, GLP-1is a 37-amino acid peptide with a MW of 4.2 KDa, having a sequencehighly conserved between different species. GLP-1 is involved inmodification of glucose homeostasis through actions that includepotentiation of glucose-stimulated insulin secretion and biosynthesisand suppression of glucagon secretion, gastric emptying, and foodintake. The abilities of GLP-1 to stimulate insulin secretion andinhibit glucagon release are glucose-dependent; thus, the risk ofhypoglycemia with GLP-1 administration is low. GLP-1 also increasesbeta-cell mass in preclinical models of diabetes through mechanisms thatinclude stimulation of beta-cell proliferation and neogenesis andinhibition of beta-cell apoptosis. Studies in both animals and humansindicate that GLP-1 may also play a protective role in thecardiovascular system.

The combined actions of GLP-1 have generated substantial interest inusing this peptide as a therapeutic agent for the treatment of type 2diabetes. However, the therapeutic potential of native GLP-1 is limitedby its very short plasma half-life (below 2 minutes). This is due toboth rapid inactivation by the proteolytic enzyme dipeptidyl peptidase(DPP)-IV and renal clearance. Consequently, long-acting,DPP-IV-resistant GLP-1 analogs have been developed for clinical use,including exenatide (Byetta, Amylin-Lilly), liraglutide (Novo Nordisk),CJC-1131 (ConjuChem), AVEO10 (Zealand Pharma-Sanofi-Aventis), LY548806(Lilly), and TH-0318 (TheraTechnologies). All these are once- ortwice-daily administration products; a controlled-release (one week)exentide product (Alkermes-Amylin-Lilly) is currently under clinicalinvestigation. These GLP-1 mimetics bind to GLP-1 receptors with similaraffinity and produce biological actions identical to those of nativeGLP-1 but are resistant to DPP-IV-mediated inactivation and renalclearance. These compounds are able to exert more sustained GLP-1-likeactivity for longer periods of time in vivo. An alternative therapeuticapproach for prolonging the action of native GLP-1 is to inhibit DPP-IVactivity, thereby preventing GLP-1 degradation. Several orally activeagents that inhibit DPP-IV activity are being evaluated for thetreatment of type 2 diabetes.

In a still further aspect, the present invention provides a kit for thepreparation of a composition of the present invention in the form of asuspension, said kit comprising at least one composition of the presentinvention in the form of a powder and optionally and preferablyinstructions for suspending the powder in an aqueous fluid.

The invention will now be further illustrated by reference to thefollowing non-limiting Examples and the attached figures, in which;

FIG. 1 shows the particle size distributions of a dispersed non-lamellarSPC/GDO/P80 sample before and after heat treatment.

FIG. 2 shows the particle size distributions of a dispersed non-lamellarSPC/GDO/P80 sample before and after heat treatment.

FIG. 3 shows cryo-transmission electron micrographs of a dispersednon-lamellar sample of SPC/GDO/P80 before and after heat treatment.

FIG. 4 shows the particle size distributions of a dispersed non-lamellarSPC/GDO/Solutol® HS 15 sample before and after heat treatment.

FIG. 5 shows a cryo-transmission electron micrograph of a dispersedsample of SPC/GDO/Solutol® HS 15 after heat treatment

FIG. 6 shows the particle size distributions of a concentrated dispersednon-lamellar SPC/GDO/P80 sample before and after heat treatment.

FIG. 7 shows the particle size distributions of a dispersed non-lamellarSPC/GDO/P80 sample after preparation and after storage for 2 months at25° C.

FIG. 8 shows the particle size distributions of a dispersed non-lamellarSPC/GDO/P80 sample after preparation and after storage for 2 months at25° C.

FIG. 9 shows the particle size distributions of dispersed non-lamellarSPC/GDO/P80 particle dispersions loaded with the anaesthetic agentPropofol at two different Propofol-to-amphiphile ratios.

FIG. 10 shows the plasma concentration of Propofol in rat afterintravenous administration.

FIG. 11 shows the particle size distribution of a dispersed non-lamellarSPC/α-tocopherol/Vitamin E TPGS sample.

FIG. 12 shows the particle size distribution of a dispersed non-lamellarEPC/GDO/P80 sample.

FIG. 13 shows cryo-transmission electron micrographs of a dispersednon-lamellar sample of SPC/GDO/P80 before and after heat treatment.

FIG. 14 shows cryo-transmission electron micrographs of a dispersednon-lamellar sample of SPC/GDO/P80 before and after heat treatment.

FIG. 15 shows the plasma concentration of Octreotide in rat aftersubcutaneous administration.

FIG. 16 shows the plasma concentration of Octreotide in rat aftersubcutaneous administration.

ABBREVIATIONS

SPC=Soy bean phosphatidylcholine from Lipoid GmbH, Germany

GDO=Glyceroldioleate from Danisco, Denmark

P80=Polysorbate 80 from Apoteket, Sweden

Solutol® HS 15=Macrogol 15 Hydroxystearate from BASF, Germany

Cryo-TEM=Cryogenic-Transmission Electron Microscopy

PPF=Propofol from Sigma-Aldrich, Sweden

EPC=Egg Phosphatidylcholine from Lipoid GmbH, Germany

DOPE-PEG(5000)=Dioleoyl phosphatidyl ethanolamine poly(ethylene glycol)5000 from Avanti Polar Lipids, U.S.A.

CMC=Carboxy Methyl Cellulose (sodium salt) from Sigma-Aldrich, Sweden

PVP=Polyvinyl pyrrolidone from ISP, U.S.A.

PEG=Polyethylene glycol from Merck, U.S.A.

EXAMPLE 1 Non-Lamellar Reversed Phase Nanoparticles

1.1—Preparation of a Non-Lamellar Dispersion

A dispersion of non-lamellar (>80% by weight of amphiphile) and lamellar(<20% by weight of amphiphile) particles, was formed by mixing 2.125 gof a SPC/GDO 40/60 wt/wt mixture (formed by mixing the lipids in ethanoland thereafter evaporating the solvent) and 0.3826 g of P80. Thecomponents were molecularly mixed by heating for 5 min at 70° C. andvortexing. The homogenous lipid melt (2.012 g) was added drop wise to38.01 g of deionized water. The resulting coarse dispersion was put on ashaking table (350 rpm) and shaken for 24 hours to give a turbidhomogenous dispersion.

The particle size was measured using laser diffraction (Coulter LS230).The size distribution was found to be narrow and monomodal with a meanparticle size of 95 nm.

1.2—Heat Treatment

An optional cycle of heat treatment was carried out on the dispersionprepared in Example 1.1.

A sample of the dispersion generated in Example 1.1 (25 mL) wasautoclaved (125° C., 20 min) and cooled to room temperature. Theparticle size distribution was narrowed, the mean particle sizeincreased to 137 nm and when examined by Cryo-TEM, a still greaterproportion of the particles showed non-lamellar character. The particlesize distribution before and after heat treatment is shown in FIG. 1.

Components:

abc aq Phase Temp Time Phase Formulation a:b:c wt % medium wt % before °C. min after i 33.9:50.8:15.3 5.0 deionized 95 Non- 125 20 Non- waterlamellar lamellar a SPC b GDO c P80

EXAMPLE 2 Further Composition

The effect of adding a higher concentration of stabilizing agent wasconsidered by preparing a second composition by the method of Examples1.1 and 1.2. A solution of SPC and GDO (40/60 wt/wt) (2.017 g) and P80(0.514 g) were molecularly mixed by heating for 5 min at 70° C. andvortexing. The homogenous lipid melt (2.006 g) was added drop wise to38.00 g of deionized water. The resulting coarse dispersion was put on ashaking table and shaken for 24 hours to give a turbid homogenousdispersion. The dispersion was thereafter heat-treated according toExample 1.2.

The size distributions before and after heat treatment were found to benarrow and monomodal with the mean particle size being 88 and 129 nm,respectively. The heat treatment also narrowed the size distribution asindicated in FIG. 2. Cryo-TEM images were obtained from the samplesbefore and after heat treatment as shown in FIG. 3. The cryo-TEM resultsclearly evidence the formation of non-lamellar nanoparticles of uniformsize containing a disordered inner structure of multiply connectedbilayers. The particles observed after heat treatment exhibit a densercore compared to those before heat treatment.

Components:

abc aq Phase Temp Time Phase Formulation a:b:c wt % medium wt % before °C. min after ii 31.9:47.8:20.3 5.0 deionized 95 Non- 125 20 Non- waterlamellar lamellar a SPC b GDO c P80

This particular composition is also well suited for preparing a liquidprecursor of the non-lamellar phase dispersion. The same components wereused in the same ratios. The components were molecularly mixed byheating to 70° C. for 5 min and vortexing. The liquid precursorformulation was also fortified with 10% by weight of a co-solvent (e.g.ethanol, N-methyl-2-pyrrolidone (NMP), propylene glycol, PEG400,glycerol) and thereafter dispersed into water (5 wt % amphiphile) withgentle shaking resulting in a milky white dispersion of non-lamellarphase particles.

EXAMPLE 3 Further Composition

The effect of adding another type of stabilizing agent was considered bypreparing another composition by the method of Examples 1.1 and 1.2. Asolution of SPC and GDO (40/60 wt/wt) (2.004 g) and Solutol® HS 15(0.516 g) were molecularly mixed by heating for 5 min at 70° C. andvortexing. The homogenous lipid melt (2.042 g) was added drop wise to38.00 g of deionized water. The resulting coarse dispersion was put on ashaking table and shaken for 24 hours to give a turbid dispersioncontaining some poorly dispersed macroscopic particles. To obtain ahomogenous dispersion, the sample was homogenised using a Microfluidizerworking at 5000 PSI and room temperature. The sample was passed throughthe homogeniser 5 times to obtain a milky homogenous dispersion. Thedispersion was thereafter heat-treated according to Example 1.2.

The size distributions obtained before and after heat treatment areshown in FIG. 4 and indicate that the heat treatment step results in amonomodal and narrow distribution with the mean particle size being 343nm. Cryo-TEM experiments after heat treatment displayed particles withdense inner non-lamellar structure as shown in FIG. 5.

Components:

abc aq Phase Temp Time Phase Formulation a:b:c wt % medium wt % before °C. min after iii 31.8:47.7:20.5 5 deionized 95 Non- 125 20 Non- waterlamellar lamellar a SPC b GDO c Solutol ® HS 15

EXAMPLE 4 Further Composition: Concentrated Non-Lamellar ParticleDispersion

A concentrated non-lamellar particle dispersion was by prepared by themethod of Examples 1.1 and 1.2. A solution of SPC and GDO (40/60 wt/wt)(4.7958 g) and P80 (0.8152 g) were molecularly mixed by heating for 5min at 70° C. and vortexing. The homogenous lipid melt (5.001 g) wasadded drop wise to 44.999 g of deionized water. The resulting coarsedispersion was put on a shaking table and shaken for 48 hours (350 rpm)to give a turbid homogenous dispersion. The dispersion was thereafterheat-treated according to Example 1.2.

The size distributions before and after heat treatment were found to benarrow and monomodal with the mean particle size being 103 and 174 nm,respectively, as indicated in FIG. 6.

Components:

abc Aq Phase Temp Time Phase Formulation a::b:c wt % medium wt % before° C. min after iv 34.2:51.3:14.5 10 deionized 90 Non- 125 20 Non- waterlamellar lamellar a SPC b GDO c P80

EXAMPLE 5 Storage Stability

Non-lamellar dispersions were prepared according to the methods ofExample 1.1 and 1.2. The compositions of the dispersions are displayedin the table below. The dispersions were stored at 25° C. and theparticle size distribution was measured at regular intervals. The sizedistributions were found to be consistent with the original sizedistribution for at least 2 months storage indicating excellentcolloidal and storage stability.

No changes of the morphology of the non-lamellar particles could beobserved (by cryo-TEM) during storage. The particle size distributionsof the original dispersions and after storage for 2 months are shown inFIG. 7 (SPC/GDO/P80=34/51/15 wt %) and 8 (SPC/GDO/P80=32/48/20 wt %). Ascan be observed, the size distributions of the original and storeddispersions are essentially identical.

Table with compositions investigated for storage stability:

Lipid concentration Composition Weight ratio (wt %) Medium SPC/GDO/P8034:51:15 5 deionized water SPC/GDO/P80 32:48:20 5 deionized water

EXAMPLE 6 Active Agent Loading

Non-lamellar particle dispersions containing the anaesthetic activeagent PPF were formed by mixing a composition comprising SPC (32% byweight of amphiphile), GDO (48% by weight of amphiphile) and P80 (20% byweight of amphiphile) with PPF at the proportions indicated in the tablebelow. The components were molecularly mixed by heating for 5 min at 70°C. and vortexing. The homogenous lipid/PPF melt was added drop wise toan aqueous solution containing 2.5% (by weight of total formulation) ofglycerol. The resulting coarse dispersions were put on a shaking table(350 rpm) and shaken for 12 hours to give homogenous dispersions. Thedispersions were thereafter heat-treated by the method of Example 1.2.The particle size distributions of the resulting dispersions were narrowand monomodal with mean particle sizes in the range of 140-150 nm asshown in FIG. 9. The PPF loaded dispersions were found to be stable tostorage at room temperature for at least 2 months.

Table with compositions of the final non-lamellar particle/PPFdispersions:

PPF:Amphiphile Amphiphile conc. (mg/mL) PPF conc. (mg/mL) (wt:wt) 50 101:5   50 20 1:2.5

EXAMPLE 7 Pharmacokinetics and Pharmacodynamics of Propofol Loaded intoNon-Lamellar Particles

A dispersion of non-lamellar particles containing PPF was prepared withthe same composition and by the same method as in Example 6 except thatthe PPF concentration in this case was 10 mg/mL and the amphiphileconcentration was 25 mg/mL (PPF:Amphiphile=1:2.5 wt/wt). Thenon-lamellar particle PPF dispersion was compared for duration ofanaesthesia in rats (male SPF Sprague-Dawley rats (Mol: SPRD HAN, M&BTaconic, Lille Skensved, Denmark)) with the reference commercialPropofol Fresenius Kabi emulsion formulation (10 mg PPF/mL). The animalswere given a single bolus intravenous injection of 10 mg PPF per kg bodyweight (induction of anaesthesia occurred directly after injection inboth cases). For pharmacodynamic parameters, the time to recover(righting response time indicated by attempts to stand up) was recorded.The results are summarized in the table below indicating the highefficiency of the non-lamellar particle PPF dispersion to maintain therequired anaesthetic effect.

Table with pharmacodynamic parameters:

Number of Average Recovering Time (sec) Formulation rats (Std. Dev.)Propofol Fresenius Kabi 5 377 (89) Non-lamellar Particle PPF 5 448 (60)Dispersion

Blood samples (0.3 mL) were collected pre-dose (one day before dosing),5 minutes, 15 minutes, 30 minutes, 1 hr, 3 hrs, 6 hrs and 24 hrs afterdosing. The Propofol concentration in rat plasma was determined by ahigh pressure liquid chromatography (HPLC) method known to scientistsskilled in the art. Plasma concentration over time of propofol wassimilar for the reference formulation and the non-lamellar particlepropofol formulation, respectively (FIG. 10). Terminal half-life(t_(half)), mean residence time (MRT), total clearance (CL),extrapolated plasma concentration at time 0 (C₀), and the totalarea-under-the-curve (AUC_(∞)) was calculated by non-compartmentalpharmacokinetic (PK) methods. AUC_(∞) was computed by the trapezoidalrule with extrapolation from C_(last) to infinity.

Table with pharmacokinetic parameters:

Formulation n t_(half (h)) MRT (h) CL (mL/h) C₀ AUC_(∞) Propofol 4 0.90(0.50) 0.88 (0.38) 4326 (1507) 2036 (329)  777 (207) Fresenius KabiNon-lamellar 5 1.97 (0.85) 2.56 (1.25) 2799 (568) 1187 (501) 1159 (243)Particle PPF Dispersion P < 0.05 (t-test) No Yes No Yes Yes

It was hypothesized that administration of propofol in the non-lamellarpropofol formulation would result in an increased circulation time inplasma. The observed PK parameters suggested that an increased presenceof propofol was indeed achieved. This was most apparent when analyzingMRT (i.e., the time any single molecule of a drug compound is residingin the circulation), which was increased approx. 3-fold compared to thereference product. Other parameters reflecting the in vivo fate ofpropofol, i.e. increased t_(half) and reduced CL for the non-lamellarparticle propofol formulation also indicated that propofol remained fora longer time in the plasma (the differences could not be statisticallyverified, but the tendency was clear). Also, AUC_(∞) increased for thenon-lamellar particle propofol formulation, implicating that theexposure to propofol was larger for this formulation than for thereference formulation at an equal dose. All observations support thatthe non-lamellar particle propofol formulation is capable of improvingthe circulation time for the active component.

EXAMPLE 8 Acute Toxicity Testing

A non-lamellar dispersion was prepared by the methods of Examples 1.1and 1.2 using the following components:

a) SPC

b) GDO

c) P80

in the weight ratio a:b:c=34:51:15, dispersed in water to a totalamphiphile concentration of 10 wt %. Sodium chloride (NaCl) was added tothe dispersion to achieve 9 mg NaCl/mL. The dispersion was thereaftertested for acute toxicity after intravenous injection in a rat model.

The non-lamellar dispersion showed no acute toxicity in a dose dependentstudy with doses up to 10 mL/kg of the 10 wt % amphiphile dispersion (1g amphiphile/kg).

EXAMPLE 9 Encapsulation of a Hydrophilic, Water-Soluble Colorant

Non-lamellar particles encapsulating the highly water-soluble colorantPatent blue were prepared as follows: 3.0 g of a formulation ofSPC/GDO/P80 (34/51/15 wt %) was prepared according to Example 1. To thissolution, 0.15 g ethanol was added and the formulation was mixed byvortex mixing. 0.20 g of an aqueous solution of Patent blue (20 mg/mL)was added to 3.0 g of the SPC/GDO/P80/EtOH formulation. The resultingsample was mixed by vortex mixing to yield a homogenous low viscosityformulation. 2.55 g of this formulation was added to 22.5 g of deionizedwater and the resulting formulation was shaken at 350 rpm for 18 hoursto give a homogenous blue-colored dispersion. After ultrafiltration(30000 MWCO filters) of the dispersion, the encapsulation efficiency wasmeasured as the absorbance (at 640 nm) of the original dispersion minusthe absorbance of the filtrate (unencapsulated fraction) and thedifference was divided by the absorbance of the original dispersion(before all absorbance measurements, TritonX100 (10 wt % in deionizedwater) was added to give clear solutions).

The encapsulation efficiency was found to be 85% indicating a highpotential of the non-lamellar particles to encapsulate water-solubleactives.

EXAMPLE 10 Encapsulation of a Hydrophilic, Water-Soluble Peptide

Non-lamellar particles encapsulating the water-soluble peptideoctreotide were prepared as follows: 1.0 g of a formulation ofSPC/GDO/P80 (34/51/15 wt %) was prepared according to Example 1. To thissolution, 0.10 g ethanol was added and the formulation was mixed byvortex mixing. 0.054 g of an aqueous solution of octreotide (35.5 mg/mL)was thereafter added and the resulting sample was mixed by vortex mixingto yield a homogenous low viscosity formulation. 1.0 g of thisformulation was added to 9.0 g of saline (9 mg NaCl/mL) and theresulting formulation was shaken at 350 rpm for 18 hours to give ahomogenous dispersion (mean particle size of ca 100 nm). Theencapsulated octreotide was separated from non-encapsulated peptide bypassing 2.5 mL of the dispersion through a Sephadex G25 (PD-10) columnand collecting the lipid fraction and the free octreotide fractions inseparate vials. The concentration of octreotide in the lipid fractionand the free octreotide fraction was analyzed, after addition ofTritonX100, by HPLC.

The encapsulation efficiency was found to be 71% again indicating thehigh potential of the non-lamellar particles to efficiently encapsulatewater-soluble actives.

EXAMPLE 11 Non-Lamellar Particles from SPC/Tocopherol Mixtures

A solution of SPC, α-tocopherol and ethanol (27/63/10 wt %) (1.34 g) wasmixed with d-α tocopheryl polyethylene glycol 1000 succinate (Vitamin ETPGS) (0.30 g). The sample was molecularly mixed by heating for 15 minat 40° C. and vortexing. The homogenous lipid melt (1.0 g) was addeddrop wise to 19 g of deionized water. The resulting coarse dispersionwas put on a shaking table and shaken for 20 hours (350 rpm) to give aturbid homogenous non-lamellar particle dispersion.

The size distribution was found to be narrow and monomodal with the meanparticle size being 128 nm, as indicated in FIG. 11.

The dispersion was thereafter heat-treated according to Example 1.2 andcryo-TEM images of the heat-treated sample displayed non-lamellarparticles containing a disordered surface structure and a dense innernon-lamellar structure.

EXAMPLE 12 The Use of EPC as a Substitute for SPC

EPC (1.539 g), GDO (2.302 g), P80 (0.685 g) was mixed with ethanol(0.501 g). The sample was mixed by vortex mixing and end-over-endrotation for 3 h resulting in a homogenous and clear liquid. The liquidformulation (1.665 g) was added to sterile water (28.335 g) and theresulting course dispersion was mixed for 18 h on a shaking table at 400rpm. The dispersion obtained was homogenous and the size distributionwas found to be narrow and monomodal with a mean particle size of 114nm, as indicated in FIG. 12.

Cryo-TEM results indicated the formation of non-lamellar nanoparticlesof uniform size containing a disordered surface structure of multiplyconnected bilayers and a dense inner non-lamellar structure.

EXAMPLE 13 Robustness of Composition

To investigate the effect of changes of the lipid composition on thefacile manufacturing of non-lamellar nanoparticles, samples with avarying ratio of SPC/GDO and a constant ratio of P80 were prepared. Thecomponents were mixed with ethanol and thereafter dispersed into sterilewater as described in Example 13 except for sample #1128 that was shakenfor 48 h. The final compositions of the samples, the mean particle sizeand polydispersity index (PI) after shaking (400 rpm) and the meanparticle size and polydispersity index after heat-treatment (accordingto Example 1.2) are indicated in the table below.

Table with compositions and data for the samples prepared in Example 14

Mean size/ Mean size/ Composition (wt %) nm PI^(a) nm PI^(a) Sample IDSPC/GDO/P80/EtOH/Water (Shaking) (Shaking) (HT^(b)) (HT^(b)) #11282.125/2.125/0.75/0.56/94.44 110 0.24 141 0.17 #11291.91/2.34/0.75/0.56/94.44 114 0.24 136 0.18 #11301.70/2.55/0.75/0.56/94.44 116 0.23 134 0.19 #11311.49/2.76/0.75/0.56/94.44 115 0.23 127 0.18 ^(a)PI = polydispersityindex, defined as the ratio between the standard deviation of the sizedistribution and the mean size; ^(b)HT = heat-treatment

From the results displayed in the table above, the non-lamellarnanoparticle system is very robust with respect to particle mean sizeand size distribution upon changes of the SPC/GDO ratio. Theheat-treatment can also be observed to narrow the particle sizedistribution (lower PI) in all cases. Cryo-TEM images of samples #1128and #1131 before and after heat treatment are shown in FIGS. 13 and 14,respectively, and display non-lamellar particles with a disorderedsurface structure of multiply connected bilayers enclosing a dense innernon-lamellar core structure.

EXAMPLE 14 Preparation of Liquid Non-Lamellar Particle Precursors

A liquid non-lamellar particle precursor was prepared by mixing SPC(1.45 g), GDO (2.15 g), P80 (0.90 g) and EtOH (0.50 g) followed byend-over-end rotation for 5 h resulting in a homogenous and clearliquid.

A second formulation was prepared by adding 2.0 g of the above liquidformulation to 0.198 g of sterile water followed by vortex mixing for 1min resulting in a clear and homogenous liquid. The exact composition ofthe liquid non-lamellar particle precursors are shown in the tablebelow.

Table with compositions of liquid non-lamellar particle precursorsSample Composition (wt %) Appearance Liquid non-lamellarSPC/GDO/P80/EtOH = Clear, homogenous particle precursor 1 29/43/18/10and light yellow liquid Liquid non-lamellar SPC/GDO/P80/EtOH/H₂O =Clear, homogenous particle precursor 2 26.4/39.1/16.4/9.1/9.0 and lightyellow liquid

The liquid precursors were readily dispensed using a syringe with a 27 Gneedle or sprayed using e.g. a pump-spray device.

EXAMPLE 15 Pharmacokinetics of Octreotide (OCT) Loaded into LiquidNon-Lamellar Particle Precursors after Subcutaneous (s.c.) Injection

A liquid non-lamellar particle precursor containing octreotide wasprepared as described in Example 15 by mixing SPC, GDO, P80, EtOH andOCT in the proportions indicated in the table below (0.6 mg octreotideper g of formulation). The resulting sample was mixed by end-over-endrotation to yield a clear and homogenous liquid formulation.

Table with composition of non-lamellar particle precursor containingoctreotide Composition (wt %) Sample ID SPC/GDO/P80/EtOH/OCT Appearance2022OCT-C 28.78/43.17/17.99/10.00/0.06 Clear, homogenous and lightyellow liquid 2022OCT-E 34.18/51.26/4.50/10.0/0.06 Clear, homogenous andlight yellow liquid

The liquid non-lamellar particle precursor formulations were injecteds.c. in rat at a dose volume of 1 mL/kg corresponding to a dose of 0.6mg OCT per kg body weight. Blood samples (0.3 mL) were collectedpre-dose (one day before dosing), 10 minutes, 30 minutes, 1 hr, 3 hrs, 6hrs, 24 hrs and 48 hrs after dosing for 2022OCT-C and pre-dose, 1 hr, 6hrs, 24 hrs, 48 hrs, 120 hrs and 168 hrs after dosing for 2022OCT-E.

The content of OCT in all plasma samples was measured by a competitiveimmunoassay. Briefly, the OCT peptide coated on a microplate competesfor the antibody in solution with the OCT present in the plasma sample.The fraction of antibody remaining in solution is removed, and thefraction bound to the immobilized peptide is quantified, the signalobtained being inversely proportional to the concentration of OCT in thesample.

The pharmacokinetics of OCT when formulated in the liquid non-lamellarparticle precursors was compared with that of a saline solution of OCT.The results in FIG. 15 show that the non-lamellar particle precursorformulations give low initial plasma levels (low “burst”) of OCT(C_(max) decreased by a factor of about 15 compared with the salinecase) and a duration of the release (or sustained release) of up to atleast 48 h for 2022OCT-C and up to at least 168 hrs (1 week) for2022OCT-E.

EXAMPLE 16 Pharmacokinetics of OCT Loaded into Non-Lamellar ParticlesDispersed in Saline after s.c. Injection

A liquid lipid stock solution was prepared by mixing SPC (0.918 g), GDO(1.377 g), P80 (0.405 g) and EtOH (0.30 g) in a glass vial followed byend-over-end rotation for 15 h. OCT (5 mg) was dissolved in sterilewater (0.095 g) and 2.2 g of the lipid stock solution was added to theaqueous octreotide solution. The resulting mixture was vortexed untilthe sample became homogenous. The lipid/octreotide mixture (1.85 g) wasadded to saline (18.15 g) and the resulting dispersion (0.2 mg OCT/mL)was mixed on a shaking table at 400 rpm for 15 h. The dispersion wasthereafter sterilized by sterile filtration (0.22 μm filter). Theresulting dispersion was turbid to milky and homogenous with a meanparticle size of ca 100 nm as measured by laser diffraction.

The liquid non-lamellar particle precursor formulations were injecteds.c. in rat at a dose volume of 3 mL/kg corresponding to a dose of 0.6mg OCT per kg body weight. Blood samples (0.3 mL) were collectedpre-dose (one day before dosing), 10 minutes, 30 minutes, 1 hr, 3 hrs, 6hrs, 24 hrs and 48 hrs after dosing.

Plasma concentration of OCT was measured as described in Example 16.

The pharmacokinetics of OCT when formulated in the non-lamellarparticles dispersed in saline was compared with that of a salinesolution of OCT. The results in FIG. 16 reveal that the non-lamellarparticle dispersion gives significantly decreased initial plasma levelsof OCT (C_(max) decreased by a factor of about 2.5 compared with thesaline case) and a duration of the release (or sustained release) of upto at least 24 h.

EXAMPLE 17 Further Compositions of OCT Loaded into Non-LamellarParticles for Injection (e.g. Intravenous (i.v.), s.c. or i.m.)

Non-lamellar particle dispersions containing OCT (0.2 mg OCT/mL) wasprepared in saline as described in Example 17. The resulting dispersionswere turbid to milky and homogenous with a mean particle size of ca 100nm as measured by laser diffraction. The compositions of theformulations are given in the table below.

Table with compositions of non-lamellar particle dispersions containingOCT Sample Composition (wt %) Appearance 2022OCT-ASPC/GDO/P80/EtOH/OCT/saline = Homogenous and2.71/4.06/1.19/0.89/0.02/91.13 white to light yellow dispersion2022OCT-B SPC/GDO/P80/DOPE-PEG(5000)/ Homogenous and EtOH/OCT/saline =white to light 2.47/4.06/1.19/0.24/0.89/0.02/91.13 yellow dispersion

The non-lamellar particle dispersions were readily dispensed using asyringe with a 31 G needle or sprayed using e.g. a pump-spray device.

EXAMPLE 18 Formulation of Salmon Calcitonin (sCT) in Liquid Non-LamellarParticle Precursors

Liquid non-lamellar particle precursors containing sCT were prepared asdescribed in Example 14 by mixing SPC, GDO, P80, EtOH and sCT in theproportions indicated in the table below (0.5 mg sCT per g offormulation). The resulting samples were mixed by end-over-end rotationto yield clear and homogenous liquid formulations.

Table with compositions of non-lamellar particle precursors containingsCT Composition (wt %) Sample ID SPC/GDO/P80/EtOH/sCT Appearance2022sCT-A 28.78/43.18/17.99/10.00/0.05 Clear, homogenous and lightyellow liquid 2022sCT-B 30.58/45.88/13.49/10.00/0.05 Clear, homogenousand light yellow liquid 2022sCT-C 32.38/48.57/9.00/10.00/0.05 Clear,homogenous and light yellow liquid 2022sCT-D 34.18/51.27/4.50/10.00/0.05Clear, homogenous and light yellow liquid

EXAMPLE 19 Freeze-Dried Powder Precursor of Non-Lamellar ParticlesContaining OCT

A liquid non-lamellar particle precursor was obtained by mixing SPC(0.3046 g), GDO (0.4570 g), P80 (0.1344 g), EtOH (0.100 g) and OCT(0.004 g) followed by end-over-end rotation for 15 h resulting in aclear homogenous liquid. The liquid precursor (0.50 g) containing OCTwas added to 9.5 g of sterile water and the resulting dispersion wasmixed on a shaking table at 400 rpm for 20 h to yield a homogenousnon-lamellar particle dispersion with 0.2 mg OCT/g of formulation. Tothe non-lamellar particle dispersion (9.0 g) was added 9.0 g of a 1 wt %aqueous solution of CMC and 18 g of a 5 wt % solution of PVP. Theresulting mixture was added to a round-bottomed flask and frozen on anEtOH/dry ice mixture followed by freeze-drying overnight. The resultingpowder was white to light yellow, of a dry consistency, contained <2 wt% of residual water and the OCT content was 1.3 mg per g of powder. Thepowder was readily redispersed in saline by vortex mixing to give amilky-white (turbid) non-lamellar particle dispersion.

EXAMPLE 20 Spray-Drying of Non-Lamellar Particles

A spray-dried non-lamellar particle precursor was obtained by mixing 6 gof a pre-made non-lamellar particle dispersion of SPC/GDO/P80 (31/54/15wt %) (5 wt % amphiphile), prepared as described in Example 1.1, with 12g of a 1 wt % aqueous solution of CMC and 12 g of a 5 wt % aqueoussolution of PVP. The resulting mixture was spray-dried using a BÜCHIMini Spray Dryer B-290 to give a white to light yellow powder with dryconsistency and <2 wt % residual water. The spray-dried powder wasreadily redispersed in saline by vortex mixing to give a milky-white(turbid) non-lamellar particle dispersion.

EXAMPLE 21 Liquid Non-Lamellar Particle Precursor and Non-LamellarParticle Dispersion Containing Insulin

A liquid lipid stock solution was prepared by mixing SPC (0.918 g), GDO(1.377 g), P80 (0.574 g) and EtOH (0.319 g) in a glass vial followed byend-over-end rotation for 15 h. Insulin (10 mg) was added to sterilewater (0.190 g) and 1.80 g of the lipid stock solution was added to theaqueous insulin solution (5 mg insulin per g of formulation). Theresulting mixture was vortexed until the sample became homogenous.

The lipid/insulin mixture (1.85 g) prepared as described above was addedto sterile water (18.15 g) and the resulting dispersion (0.46 mginsulin/mL) was mixed on a shaking table at 400 rpm for 15 h. Theresulting dispersion was turbid to milky and homogenous.

EXAMPLE 22 Liquid Non-Lamellar Particle Precursor and Non-LamellarParticle Dispersion Containing GLP-1

A liquid lipid stock solution was prepared by mixing SPC (0.918 g), GDO(1.377 g), P80 (0.574 g) and EtOH (0.319 g) in a glass vial followed byend-over-end rotation for 15 h. GLP-1 (10 mg) was added to sterile water(0.190 g) and 1.80 g of the lipid stock solution was added to theaqueous insulin solution (5 mg insulin per g of formulation). Theresulting mixture was vortexed until the sample became homogenous.

The lipid/GLP-1 mixture (1.85 g) prepared as described above was addedto sterile water (18.15 g) and the resulting dispersion (0.46 mgGLP-1/mL) was mixed on a shaking table at 400 rpm for 15 h. Theresulting dispersion was turbid to milky and homogenous.

The invention claimed is:
 1. A particulate composition comprising; a) 5to 50% of at least one phosphatidyl choline component b) 5 to 90% of atleast one diacyl glycerol component, at least one tocopherol, ormixtures thereof, and c) 1 to 40% of at least one non-ionic stabilisingamphiphile consisting essentially of surfactants having a molecularweight below 8000 amu, wherein all parts are by weight relative to thesum of the weights of a+b+c and wherein the composition comprisesparticles of at least one non-lamellar phase structure or formsparticles of at least one non-lamellar phase structure when contactedwith an aqueous fluid; wherein, when contacted with an aqueous fluid toform particles having a non-lamellar phase structure, no more than 1% ofparticles have a particle size outside the range of 0.05-1.5 μm; andwherein the compositions are essentially stable in terms of phasebehaviour, particle size and particle size distribution for at least 3months.
 2. The composition as claimed in claim 1 wherein component a)comprises at least one PC selected from Egg PC, Heart PC, Brain PC,Liver PC and Soy PC.
 3. The composition as claimed in claim 1 whereincomponent b) comprises a diacyl glycerol having acyl chains with 14 to18 carbons.
 4. A composition as claimed in claim 1 wherein component b)is GDO, or a mixture of tocopherol with GDO.
 5. The composition asclaimed in claim 1 wherein component a) and/or component b) are derivedfrom a natural source.
 6. The composition as claimed in claim 5 whereincomponent a) has at least 50% C18:1 and/or C18:2 acyl groups andcomponent b) is a diacyl glycerol with at least 50% C18:1 and/or C18:2acyl groups.
 7. The composition as claimed in claim 1 wherein componentc) comprises at least one non-ionic stabilising amphiphile selected fromare Polysorbates 20 and 80, solutol, d-alpha tocopheryl polyethyleneglycol 1000 succinate (Vitamin E TPGS) and polyethoxylated caster oil.8. The composition as claimed in claim 1 additionally comprising anactive agent.
 9. The composition as claimed in claim 8 wherein saidactive agent is at least one selected from octreotide and othersomatostatin related peptides, insulin, chlorhexidine digluconate,chlorhexidine dihydrochloride, bisphosphonates, non-steroidalanti-inflammatories, corticosteroids, methotrexate, azathioprine,6-mercaptopurine and phospholipase inhibitors.
 10. The composition asclaimed in claim 5 wherein the composition comprises particles of I₂and/or L₂ phase structure and/or forms particles of I₂ and/or L₂ phasestructure when contacted with an aqueous fluid.
 11. The composition asclaimed in claim 1 wherein the average particle size of the particlescomprising the composition or formed on contact with an aqueous fluid is0.1 to 0.6 μm.
 12. The composition as claimed in claim 1 furthercomprising up to 20% of at least one organic solvent having 1 to 6carbon atoms and/or water-soluble polymers thereof.
 13. The compositionof claim 1 in the form of: a) a dispersion b) a pre-concentrate in aco-solvent, or c) a dry powder, or a solidified mixture with abiotolerable polymer.
 14. A pharmaceutical formulation comprising atleast one composition as claimed in claim 5 and at least onebiologically tolerable carrier or excipient.
 15. The pharmaceuticalformulation as claimed in claim 14 in a form selected from suspensionsin liquid, powders, tablets, capsules, coated capsules, coated tablets,aerosols, suppositories, drops, creams, transdermal patches and sprays.16. A formulation as claimed in claim 15, which is suitable forparenteral administration.
 17. The composition as claimed in claim 1wherein the weight ratio of (a):(b) ranges from 1:5 to 3:2.
 18. Thecomposition as claimed in claim 1 wherein both PC and DAG componentscomprise at least 50% oleoyl or linoleoyl acyl groups.
 19. A method forthe formation of non-lamellar particles comprising forming a mixturecomprising; a) 5 to 90% of at least one phosphatidyl choline componentb) 5 to 90% of at least one diacyl glycerol component, at least onetocopherol, or mixtures thereof, and c) 2 to 40% of at least onenon-ionic stabilising amphiphile consisting essentially of surfactantshaving a molecular weight below 8000 amu, wherein all parts are byweight relative to the sum of the weights of a+b+c and dispersing saidmixture in an aqueous fluid and wherein the composition comprisesparticles of at least one non-lamellar phase structure or formsparticles of at least one non-lamellar phase structure when contactedwith an aqueous fluid; wherein, when contacted with an aqueous fluid toform particles having a non-lamellar phase structure, no more than 1% ofparticles have a particle size outside the range of 0.05-1.5 μm; andwherein the compositions are essentially stable in terms of phasebehaviour, particle size and particle size distribution for at least 3months.
 20. A kit for the preparation of a composition in the form of asuspension, said kit comprising at least one particular composition ofclaim 1 in the form of a powder and optionally instructions forsuspending the powder in an aqueous fluid.
 21. A method for thetreatment of a human or animal subject comprising administration of acomposition as claimed in claim
 1. 22. The method of treatment asclaimed in claim 21 for the treatment of inflammation and/or irritation,in a body cavity.
 23. The method as claimed in claim 21 for thetreatment of inflammatory bowel disease.
 24. A method for the sustainedrelease of an active agent over a period of 1 to 30 days comprisingadministering a formulation comprising a composition of claim 5 and atleast one active agent.